Familial Mediterranean fever (FMF) is an inherited idsorder of inflammation characterized by episodes of fever with abdominal pain, pleurisy, and/or arthritis; some patients also develop systemic amyloidosis, leading eventually to renal failure. FMF is caused by one or more recessive mutations at a locus (designated MEFV) on chromosome 16p. The carrier frequency for FMF is very high among Jewish, Armenian, Turkish, and Arab populations. Since the biochemical lesion in FMF is unknown, we have undertaken a positional cloning strategy to identify this gene and its mutations. By the beginning of this year we had narrowed the candidate interval to a region of approximately 375 kb. In 1995-96 our efforts were divided among a) completing the genomic cloning of the candidate interval; b) further genetic refinement of the interval; c) assemblage of a transcriptional map of the interval; and d) mutational analysis of positional candidate genes. We have now completed the genomic cloning and high-resolution restriction mapping of a l,050 kbregion that contains the refined MEFV candidate interval. Our map consists of 179 cosmid, 15 P1, 6 PAC, 3 BAC, and 19 YAC clones, anchored by 30 STS markers generated from either end-sequencing of clones or by microsatellite screening. Notl and EcoRI mapping of the overlapping cosmids, hybridization of restriction fragments from cosmids to one another, and STS analyses have been used to validate the contig. From these genomic clones we have identified five new polymorphic markers: two are AC repeats, one is an ATT repeat, and two others are single-nucleotide polymorphisms. Based on historical recombinants in a North African Jewish founder chromosome, we have further refined the candidate interval to 240 kb. Moreover, we have assembled a transcriptional map of the candidate interval, using direct cDNA selection, exon amplification, BLAST analysis of single-pass sequence data, and hybridization of labeled cosmid inserts to arrayed cDNA libraries. To date we have identified parts of 16 apparently independent transcripts, including one leukocyte activation molecule, a member of the olfactory receptor family, and at least 3 genes with zinc-finger motifs. Current experiments are directed towards identifying full-length transcripts from partial cDNAs and exons, and mutational screening. Three full-length genes have already been sequenced in a panel of 8 individuals (5 affected, 2 normal, 1 carrier), without finding a disease-associated mutation. During the next year we will continue transcript-extension and mutation-detection until we identify a disease-associated gene. Subsequent experiments will address molecular and biochemical events in the pathophysiology of FMF.